Taken as a whole, cancers are a significant source of mortality and morbidity in the U.S. and throughout the world. However, cancers are a large and varied class of diseases with diverse etiologies. Researchers therefore have been unable to develop treatments or diagnostic tests which cover more than a few types of cancer.
For example, cancers are associated with many different classes of chromosomal features. One such class of chromosomal features are perturbations in the genomic structure of certain genes, such as the deletion or mutation of tumor suppressor genes. The activation of proto-oncogenes by gene amplification or promoter activation (e.g., by viral integration), epigenetic modifications (e.g., a change in DNA methylation) and chromosomal translocations can also cause cancerigenesis. Such perturbations in the genomic structure which are involved in the etiology of cancers are called “cancer-associated genomic regions” or “CAGRs.”
Chromosomal fragile sites are another class of chromosomal feature implicated in the etiology of cancers. Chromosomal fragile sites are regions of genomic DNA which show an abnormally high occurrence of gaps or breaks when DNA synthesis is perturbed during metaphase. These fragile sites are categorized as “rare” or “common.” As their name suggests, rare fragile sites are uncommon. Such sites are associated with di- or tri-nucleotide repeats, can be induced in metaphase chromosomes by folic acid deficiency, and segregate in a Mendelian manner. An exemplary rare fragile site is the Fragile X site.
Common fragile sites are revealed when cells are grown in the presence of aphidocolin or 5-azacytidine, which inhibit DNA polymerase. At least eighty-nine common fragile sites have been identified, and at least one such site is found on every human chromosome. Thus, while their function is poorly understood, common fragile sites represent a basic component of the human chromosome structure.
Induction of fragile sites in vitro leads to increased sister-chromatid exchange and a high rate of chromosomal deletions, amplifications and translocations, while fragile sites have been colocalized with chromosome breakpoints in vivo. Also, most common fragile sites studied in tumor cells contain large, intra-locus deletions or translocations, and a number of tumors have been identified with deletions in multiple fragile sites. Chromosomal fragile sites are therefore mechanistically involved in producing many of the chromosomal lesions commonly seen in cancer cells.
All malignant cells have specific alterations at DNA loci that encode genes for oncoproteins or tumor suppressors (Balmain et al., 2003; Wooster and Weber, 2003). This common feature has recently been expanded to include a large class of non-coding RNAs (ncRNAs) called microRNAs (miRNAs) (Ambros, 2004) that are also involved in cancer initiation and progression (Calin et al., 2002; Croce and Cann, 2005; Berezikov and Plasterk, 2005a; Esquela-Kerscher and Slack, 2006; Calin and Croce, 2006a). mRNAs affect the regulation of gene expression at both the transcriptional and post-transcriptional levels (Ambros, 2003; Ambros, 2004).
The extent of involvement of miRNAs and the involvement of other classes of ncRNAs in human tumorigenesis is unknown. Therefore, there is a need for further research into the molecular mechanisms and signal transduction pathways altered in cancer.
There is a further need for the identification of new molecular markers and potential therapeutic agents.
The ultraconserved regions (UCRs) of the human genome (Bejerano et al., 2004b) are also miRNAs that are almost completely conserved among various species (Berezikov et al., 2005b). For example, the active molecules of the miR-16-1/miR-15a cluster, has been shown to be an essential player in the initiation of chronic lymphocytic leukemia (CLL) (Calin et al., 2005a), and are completely conserved in human, mouse and rat and highly conserved in nine out of the ten sequenced primate species (Berezikov et al., 2005b). Comparative sequence analysis has identified a number of highly conserved genomic sequences. Some of these regions do not produce a transcript that is translated into protein and are therefore considered to be non-genic. Various names have been applied to this class of sequences: conserved non-genic sequences (CNGs) (Dermitzakis et al., 2005), conserved non-coding sequences (CNSs/CNCs) (Meisler, 2001), multiple species conserved sequences (MCSs) (Thomas et al., 2003) or highly conserved regions (HCRs) (Duret et al., 1993).
UCRs are a subset of conserved sequences that are located in both intra- and inter-genic regions. They are absolutely conserved (100%) between orthologous regions of the human, rat, and mouse genomes (Bejerano et al., 2004b). In contrast to other regions of conserved sequence, 53% of the UCRs have been classified as non-exonic (‘N’, 256/481 without evidence of encoding protein), while the other 47% have been designated either exonic (‘E’, 111/481, that overlap mRNA of known protein-coding genes), or possibly exonic (‘P’, 114/481, with inconclusive evidence of overlap with protein coding genes).
A large portion of transcription products of the non-coding functional genomic regions have significant RNA secondary structures and are components of clusters containing other sequences with functional non-coding significance (Bejerano et al., 2004a). The UCRs represent a small fraction of the human genome that are likely to be functional but not encoding proteins, and have been called the “dark matter” of the human genome (Bejerano et al., 2004a). Because of the high degree of conservation, the UCRs may have fundamental functional importance for the ontogeny and phylogeny of mammals and other vertebrates. This was illustrated by the recent finding of a distal enhancer and an ultraconserved exon derived from a novel retroposon active in lobe-finned fishes and terrestrial vertebrates more than 400 million years ago and maintained as active in a “living fossil” coelacanth (Bejerano et al., 2006).
Further experimental proof of the functional importance of UCRs is based on analysis of mice with targeted mutations. Megabase deletions of gene deserts that lack ultraconserved elements or highly conserved sequences resulted in viable mice that developed apparently without detectable phenotypes (Nobrega et al., 2004). By contrast, gene deserts containing several UCRs (such as the two gene deserts surrounding the DA CHI gene on human chromosome 13g21.33) were shown to contain long-range enhancers, some of them composed of UCR sequences (Nobrega et al., 2003).
In spite of considerable research into therapies for cancer-related diseases, these diseases remain difficult to diagnose and treat effectively. Accordingly, there is a need in the art for improved methods for diagnosing and/or treating cancer. The present invention fulfills these needs and further provides other related advantages.